Project Abstract Non-canonical miRNA biogenesis mechanisms in Drosophila and mammals microRNAs (miRNAs) are an abundant class of small regulatory RNAs that derive from consecutive cleavages of hairpin transcripts by the Drosha and Dicer RNase III enzymes. The resultant mature miRNAs mediate extensive networks of post- transcriptional regulation, and are implicated in a variety of diseases including cancer. In the course of analyzing the miRNA pathway in Drosophila, we uncovered a new class of miRNA derived from short hairpin introns that we term mirtrons. These atypical substrates bypass Drosha to generate functional miRNAs, and we later showed that splicing-derived miRNAs also exist in mammals. Certain individual miRNAs, including most famously the let-7 tumor suppressor miRNA, are regulated by terminal uridylation. We found that mirtrons as an entire class are subject to specific and abundant uridylation-mediated repression at the hairpin stage. In particular, we (1) recently elucidated and dissected the Drosophila mirtron terminal uridyltransferase (TUTase) system, (2) discovered an immense proliferation in the numbers and biogenesis strategies for mirtrons in mammals, (3) find mirtron uridylation to be broadly conserved in mammals. We now propose to elucidate the molecular features, functional impacts and evolutionary bearing of TUTase systems that target splicing-mediated miRNA pathways in mammals. Beyond this, we provide compelling evidence for additional requirements for Drosophila TUTases and their protein partners that extend the reach of these RNA modification systems into new regulatory pathways. These preliminary data are the basis of an integrated set of biochemical, molecular, genetic and genomewide studies to reveal novel TUTase mechanisms and biological functions. Our efforts to understand these questions will eventually further our knowledge of how to control the activity of small regulatory RNAs in humans.